JP4599588B2 - Cell manipulation method using cell picking tool comprising molded article with cell adhesive concave structure - Google Patents

Description

The present invention relates to a cell manipulation method using a molded body having a concave structure capable of collecting a cell sheet that has been two-dimensionally cultured in a culture vessel, without using a cell dispersing agent, in the form of a sheet, More specifically, by placing it in contact with the cell aggregate in the cell growth environment, the liquid component (cell growth environment) that contributes to cell growth is taken in, and the cells are formed in a concave structure while maintaining the assembled state. The present invention relates to a cell manipulation method using a cell collection molded body (cell picking tool) that can be collected.

The present invention relates to a method for culturing living cells and the use of cultured cells produced by the method. For example, cells cultured on a petri dish are peeled off with a cell peeling agent such as trypsin as in the conventional method. Can be collected without the need for a process to be performed, and by moving the molded body from which the cells have been collected, the cells can be seeded and passaged in an arbitrary cell growth environment. The complexed cells can be made into a two-dimensional or three-dimensional aggregate by a simple operation, and further, different cells are accumulated in one place together with a molded body having a suitable growth environment. that can be useful new cells operating method having the excellent features not in the conventional method etc. as to provide Hisage.

The cell manipulation method using the cell picking tool of the present invention provides, for example, a new cell manipulation technique in the field of cell research that contributes to medical technology and genome science. For example, minimally invasive cell collection / passaging, 3 It can be suitably used for new cell manipulation methods in dimensional cell culture, cell therapy, co-culture. In this specification, the cell manipulation method may be referred to as a cell handling manipulation method or a cell handling method.

  Cell culture technology that has been accumulated as a basic technology supporting biotechnology is a powerful tool for cell manipulation in the fields of tissue engineering, regenerative medicine, and drug discovery, for example. Is often desired to be in a handleable sheet or agglomerated form. Today, the most commonly used cell culture mode is two-dimensional culture on a culture vessel such as a culture dish or petri dish. In the two-dimensional culture system, the cells adhere to the bottom surface of the culture container, which is advantageous in terms of proliferation, while the cultured cells are strongly bound to the culture container. Therefore, when the cells cultured in the two-dimensional culture system are passaged and operated, it is essential to detach the cells from the culture vessel by treatment with a cell detachment agent such as trypsin treatment. However, according to the above method, the cultured cells have a problem that they cannot be damaged by the cell peeling agent. Further, in the above method, since the extracellular matrix (ECM) is lost, it is extremely difficult to recover the cultured cells in a useful form (a sheet form or a lump form) and maintain their cell differentiation ability. There is a problem.

  As a conventional method for solving the above-mentioned problems, for example, there is a method (for example, Non-Patent Document 1, Patent Document 1) in which suspended cells are attached to a microcarrier surface and cultured. However, in the above method, the process of making the cultured cells into suspension is inevitable, and since the cells are not subject to physical stimulation (collision with the container or handling), many cells die. Moreover, since the microcarrier surface is convex, it is difficult to finish cells into a high-density aggregate. As another method, for example, a method in which cells are encapsulated with a gel such as calcium alginate and cultured (for example, Patent Document 2) has been reported. However, even in the above-described method, the process of making the cultured cells into suspension is inevitable, and many cells die during complicated steps of encapsulation. In addition, since the gel capsule completely covers the cells, sufficient gas exchange cannot be performed, and long-term culture is difficult. Furthermore, handling is difficult due to insufficient strength of the gel capsule.

  In recent years, particularly in the field of regenerative medicine, there has been a strong demand for a method for recovering cultured cells in a useful form (a sheet or a lump). As a technique for answering the above requirement, a method of floating culture of cells on a cell non-adhesive substance or a method of agglutinating cells that have been naturally detached from a weakly adherent substrate has been reported. In addition, in relation to the above method, a method of culturing cells on a poly (N-isopropyl acrylamide) thin layer that contracts at a temperature of 32 ° C. or higher, heating the cells when they become confluent, and reporting the sheet-like cells has been reported. (For example, Non-Patent Document 2). In addition, a method for obtaining high-density cell aggregates by repeating cell pelletization by centrifugation and suspension culture of pelleted cells has been studied (for example, Non-Patent Document 3). However, the cell sheet and the cell aggregate produced by the above method are very delicate and cannot withstand clinical applications that require handling with tweezers, for example.

JP 59-67965 A JP-A-10-248557 L. Ikonomouet al., BIOTECHNOLOGY PROGRESS 18 (6), p.1345-1355 NOV-DEC 2002 T. Okano et al., J. Biomed. Mater. Res., Vol. 27, p. 1243-1251, 1993 Y. Kato et al., Proc. Natl. Acad. Sci. U.S.A., 85, 9552, 1988

Under such circumstances, the present inventor, in view of the above-described prior art, has diversified a new cell manipulation tool that can surely solve the problems in the prior art, a new form of use thereof, and a product thereof. As a result of investigating from various viewpoints and earnest research, we adopted a cell manipulation method using a molded body with a concave structure that can collect part or all of cultured cells, cell aggregates, and biological tissues together with a predetermined cell growth environment As a result, it has been found that the intended purpose can be achieved, and the present invention has been completed.

That is, the present invention provides a cultured cell in an appropriate culture environment, taking transferred to each culture environment moldings, using cells harvested for moldings which can be moved in the culture environment of the molding for different purposes (cell picking tool) The object is to provide a cell manipulation method . Another object of the present invention is to provide a method for subculturing cells cultured in a culture container such as on a petri dish in another culture environment using the cell picking tool. The present invention also provides a method for co-culturing a plurality of cultured cells by moving and placing them in a single-purpose culture environment for each cell picking tool that holds each culture environment. It is for the purpose.

Another object of the present invention is to provide a method for forming an appropriate cell agglomerate using, for example, a concave structure of a molded body that maintains a cell growth environment. Furthermore, the present invention provides an application in which the cell aggregate formed in the concave structure of the molded body is used as an injection / filler for a living body together with the molded body, for example, when the molded body has a material structure that is not harmful to the living body It is possible to do.

In order to solve the above-mentioned problems, the present invention provides a cell picking tool comprising a molded article having a cell adhesive concave structure in a cell collection site of a container during cell culture, thereby allowing the cells to adhere and proliferate on the molded article side. And a cell manipulation method comprising conducting passive cell collection and operating the cell picking tool for handling, wherein (a) the compact is a calcium phosphate ceramic, (b) the compact is It is a shape having a cross section with an aspect ratio (long axis / short axis) of 1.005 to 5, and when left on a flat surface, part or all of the pores, through holes, or dimple openings face downward. (c) the concave structure is composed of a material having a cellular contact adhesion, an opening area of (d) concave structure, a 100~9 × 10 ⁶ μm ^2, and (e) concave structure opening Cell sea When are in contact, concave structure opening and the cell sheet is adhered, it Ru is characterized by using the cell picking tool, a cell sheet which is two-dimensionally cultured in a culture vessel, without using a cell dispersing agent The cell manipulation method is characterized in that the cells are collected in a sheet form while maintaining the bound state of the cells . This procedure, (1) formed form is from ^{5 × 10 -4 1 × 10 3} mm 3, spherical, bead-like, massive, plate-like, polyhedral, burr-like, dendritic, and the organic projecting shapes ( 1 ) the molded body is a pore, a through-hole, a dimple, a slit, a node formed by a projection coupling portion, a surface adsorbed protein, a hydrophilic treatment It is a preferred embodiment that the molded body has one or two or more structures selected from the group consisting of a surface, a polymer coat, and an oxide film .

Next, the present invention will be described in more detail.
In the present invention, the molded body contains, for example, calcium phosphate ceramics (for example, hydroxyapatite, β-TCP, etc.) and single crystals thereof, polystyrene, collagen gel, gelatin, sodium alginate gel, calcium phosphate at an appropriate concentration. Agar is preferable from the viewpoint of cell collection, but is not limited thereto, and if it is substantially the same as these or similar, use them in the same way. Can do. Opening area of the concave structure of the molded body to be 100~9 × 10 ⁶ μm ^2, maintenance of cell sheet structure, is required from the viewpoint of achieving both the retention and cell infiltration liquid component. Moreover, molded bodies, for example, be from 5 × 10 ^-4 is 1 × 10 ³ mm ³ about spherical particles, it is preferred from the viewpoint of handling. Further, when the molded body is allowed to stand on the cell growth environment plane, a part of the concave structure opening surface of the molded body or a part or all of the concave structure opening of the molded body is in contact with the cell growth plane or It is necessary to make everything almost perpendicular to the short axis of the compact.

  The collection of cells using the molded body is performed by introducing the molded body into a cell collection site such as a culture dish. The shaped body is put into a cell collection site in a sterilized state. Examples of sterilization methods include, but are not limited to, autoclave sterilization, gas sterilization, dry heat sterilization, and ultraviolet sterilization. Anything can be used as well. As described above, the cell collection site into which the molded body has been put is placed in an incubator set to an appropriate environment. In the above operation, the indwelling period may be appropriate, but it is preferably 6 to 240 hours. The molded body put in the cell collection site in the culture dish is formed from a liquid component (cell growth environment) that contributes to cell growth of the cell collection site, for example, a medium component of serum-containing medium, serum. It is taken into the body surface and inside, and comes into contact with cells (FIG. 1). Cells in contact with the molded body adhere to the molded body surface as a scaffold and are collected on the molded body (FIG. 2) (passive cell collection by the molded body). At this time, in the concave structure, when the opening has a predetermined area, the cells are collected while maintaining the assembled state. The cells collected in the micromolded body continue to grow and reach confluent. In particular, in the concave structure of the micro-molded product, the cells proliferate so as to fill the concave structure and form cell aggregates (FIG. 3).

In the present invention, the cell is preferably a cell aggregate (cell collection site) in a culture vessel during cell culture, for example, any sub-confluent to confluent, culture in a two-dimensionally cultured culture dish. It is collected from a cell aggregate composed of 1 × 10 ⁴ or more cells in a dish and the surface of a cell sheet formed in a suspension culture system. The culture vessel is suitable in terms of cell growth. However, the present invention is not limited to these, and any one having substantially the same effect as these or similar to these can be used in the same manner. Each of the above cell collection sites includes a medium suitable for the cells. In the medium, for example, cells of about 1 × 10 ⁶ cells / ml may be suspended as necessary. In the present invention, cells are collected from one or two or more cell collection sites selected from the above.

Cells collected in the molded body leak out of the molded body during the growth process (FIG. 4). At this time, when the molded bodies are adjacent to each other, the molded bodies are cross-linked by the proliferated cells and can maintain an arbitrary structure. That is, fine胞採collector compacts (cell picking tool) is preferably, for example, shows the cell adhesion properties, the molded article having a concave structure which can achieve both the maintenance and liquid component holding cell sheet structure When the concave structure opening of the cell picking tool and the cell sheet are in contact with each other, it exhibits a function of bonding the concave structure opening and the cell sheet. Therefore, by placing the sterilized cell picking tool in the cell collection site, the cell growing environment of the cell collection site and the cells can be infiltrated / proliferated in the cell picking tool. By collecting the cell picking tool in which cells have infiltrated and proliferated and moving to another culture environment, the cells collected in the cell picking tool leak at the destination, so that any cell can be targeted. It can be seeded and subcultured in a culture environment. In addition, arbitrary cells collected by the cell picking tool are assembled three-dimensionally together with the cell picking tool, whereby a three-dimensional cell culture system and a cell aggregate having a three-dimensional cell structure can be obtained. Furthermore, in the present invention, for example, when the cell picking tool has a material configuration that is not harmful to the living body, the cell aggregate formed in the concave structure of the cell picking tool is used as the injecting / filling agent for the living body together with the cell picking tool. be able to. However, the present invention is not limited to these methods.

  The present invention passively collects cells cultured by an appropriate method, for example, a two-dimensional culture method using a culture dish advantageous for proliferation, to efficiently and effectively culture cells. It is applied to various uses. According to the conventional method, the cultured cells are once detached from the culture container with a cell peeling agent such as trypsin, adjusted to a cell suspension, and applied to various uses. However, the cultured cells collected with the cell detachment agent almost lose the extracellular matrix (ECM) that contributes to cell growth / differentiation and the like, and are not suitable for application to, for example, cell therapy and tissue engineering. On the other hand, according to the present invention, cultured cells can be collected into a molded body that can be handled without using a cell release agent. The method of the present invention that does not use a cell detaching agent is minimally invasive to cells, and the obtained cultured cells retain abundant useful ECM. Furthermore, according to the present invention, since the cells collected in the molded body continue to grow in the molded body, a high-density cell aggregate that survives for a long period of time can be produced.

  The cells collected in the molded body can move together with the molded body. In particular, in the cells collected in the through-holes or the concave structure of the molded body, there is no damage associated with handling by tweezers or the like as in the conventional method. Moreover, since the molded body holds liquid components necessary for cell growth, the cells are not dried during movement. Therefore, according to the present invention, cultured cells can be collected in a form useful for cell therapy, tissue engineering and the like. Moreover, a three-dimensional cell culture system and a three-dimensional structure cultured cell can be assembled by assembling the molded body from which the cells are collected into a desired three-dimensional structure.

In the present invention , by making full use of a cell picking tool, for example, by moving a molded body from which cultured cells are collected to a new petri dish, cell passage and seeding can be performed in a less invasive manner. Further, different cells cultured in an appropriate culture methods, harvested by cells picking tool, by moving to any culture environment, it is possible to perform co-culture (co-culture). Furthermore, when the cell picking tool has a material configuration that is not harmful to the living body, the cell picking tool obtained by collecting the cells constituting the tissue to be regenerated can be reliably placed in the treatment target region to back up the tissue regeneration ( Cell therapy). For example, when bone cells or cells that may be differentiated into bone are collected in a calcium phosphate molded body (such as hydroxyapatite), they can be used as an injection for hard tissue regeneration.

In the present invention, the cell picking tool, Ru commercialization possible der this as a kit of sterile packaging. For example, a cell picking tool can be packed in an appropriate bag or capsule space to prepare a filling, sterilized and packed, and combined with an appropriate cell culture environment to obtain a predetermined product. In this case, the cell picking tool can be mixed with an appropriate cell culture environment to form a packing material. In the present invention, the cell picking tool can be loaded with an arbitrary drug component to form a filling material. Examples of these optional drug components include antibiotics, anti-inflammatory agents, platelet-rich plasma, and BMP. However, it is not limited to these, and an appropriate drug component can be carried.

In the present invention, a molded body is placed in a cell aggregate (cell collection site) in a cell culture vessel during cell growth, thereby incorporating a liquid component (cell growth environment) that contributes to cell growth into the molded body. Cell manipulation using a new cell collection medium that allows cells to be handled together with the molded body using the molded body-cell complex obtained by adhering and proliferating to the molded body side (passive cell collection) According to the present invention, according to the present invention, (1) cultured cells can be collected, transferred, and seeded without using a cell detachment agent such as trypsin. (2) Cell collection using the cell collection medium described above, It is minimally invasive to cells, (3) cell passage can be easily performed by making full use of the cell collection medium, and (4) a three-dimensional structure can be made by making full use of the cell collection medium. (5) By making full use of the cell collection medium, a plurality of cultured cells can be collected in one arbitrary place and co-cultured ( 6) The cell aggregate formed in the cell collection medium can be handled intuitively, and exhibits an exceptional effect that it is useful as a biomedical injection / filler for regenerative medicine.

  EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited at all by the following Examples.

  Hydroxyapatite (HA) powder prepared to a particle size of 50 μm or less was mixed with 1 wt% sodium alginate aqueous solution so as to be 20 wt% to obtain a uniform slurry. The slurry was dropped into a 1 wt% calcium chloride aqueous solution to form an HA gel sphere having a diameter of 1.6 mm. The size of the apatite gel sphere was controlled by dropping the slurry with a digital pipette. Before the HA gel sphere was dried, a through-hole passing through the center of the HA gel sphere was drilled with a stainless steel wire of φ500 μm. By this work, the HA gel sphere could be flattened in the direction of the through hole. The HA gel sphere after the formation of the through hole was dried at 60 ° C. for 12 hours and then sintered at 1200 ° C. for 1 hour. HA ceramic beads (cell picking tool, HAB) having a major axis of 1 mm and a minor axis of 0.9 mm (aspect ratio of 1.11) having 300 μm through holes were produced. The prepared HAB was stable on the plane with the through hole opening facing the bottom surface (FIG. 5).

The HAB prepared in Example 1 was subjected to ultrasonic cleaning for 10 minutes in 99.5% ethanol. Human osteosarcoma cells MG63 were cultured in a 6-well (diameter 9.6 cm ² / well) culture dish to obtain a subconfluent state. In the above culture, Dulbecco's MEM + 10% FBS + 1% P.I. S. Was used as the medium. HAB sterilized by dry heat at 200 ° C. for 2 hours was put into the above culture dish at 20 pieces / well and left in an incubator at 37 ° C. and 5% CO ₂ . Through the above operation, MG63 on the culture dish could be collected in HAB (FIG. 6). The amount of MG63 collected into HAB increased in proportion to the standing period, and became 537, 2970, and 4728 cells / HAB after 24, 72, and 120 hours, respectively (FIG. 7). The amount of MG63 collected in the molded body HAB was determined from the amount of DNA extracted from cells in HAB. Viability (viable cell rate) of MG63 collected in HAB was 95% or more. HAB collected from MG63 in a stationary period of 1 day was transferred to a 12-well culture dish and Dulbecco's MEM + 10% FBS + 1% P.I. S. Was left as a medium in an incubator. Through the above operation, MG63 could be seeded in a 12-well culture dish (FIG. 8). After this, MG63 aggregates could be formed in the HAB through holes.

  Sixty-six HABs collected in Example 2 from which MG63 was collected were moved to a 96-well culture dish, HABs were three-dimensionally arranged, and left in an incubator for 48 hours. As a result, adjacent HABs could be cross-linked with the grown MG63.

  A HAB obtained by collecting the mouse osteoblast cell line MC3T3-E1 was prepared in the same manner as in Example 2, except that the mouse osteoblast cell line MC3T3-E1 was used instead of MG63. The HAB from which MC3T3-E1 was collected and the HAB from which MG63 was produced in Example 2 were placed on a 12-well culture dish so that eight of them were alternately arranged. As a result, a coculture system in which MC3T3-E1 and MG63 were seeded in an alternating arrangement could be produced.

The present invention relates to a cell manipulation method using a molded article having a concave structure that exhibits cell adhesion properties and can maintain both the maintenance of the cell sheet structure and the retention of liquid components. The cells cultured in the cell collection site can be collected without detaching with a cell detachment agent, that is, while maintaining the cell aggregation state, and the cells can be seeded and passaged in an arbitrary cell growth environment. The method of the present invention is less invasive to cells and can operate cultured cells without degrading useful ECM. Further, according to the present invention, by assembling the molded body, the cells combined with the molded body can be made into a two-dimensional or three-dimensional aggregate. Furthermore, according to the present invention, for example, different cells can be collected and cultured in one place together with the shaped bodies that have a suitable growth environment. The cell handling system of the present invention is useful as a device that can realize minimally invasive cell collection / passaging, three-dimensional cell culture, cell therapy, co-culture method, and the like. According to the present invention, for example, a new technique for cell manipulation in the fields of tissue engineering, regenerative medicine, and drug discovery can be provided.

The model is put into a cell collection site in a culture dish, a medium component containing serum is taken into the surface and inside thereof, and a schematic view of the state in contact with the cultured cells is shown. The schematic diagram of a state in which cells in contact with the molded body adhere to the opening of the concave structure of the molded body and are collected while maintaining the cell sheet structure (passive cell collection by the molded body) is shown. The schematic diagram of the state which the cell extract | collected by the molded object continued proliferation, reached to confluent, and came to form a cell aggregate is shown. The schematic diagram of a mode that the cell extract | collected in the molded object leaks out of a molded object is shown. An example of the optical microscope photograph of HA molded object (cell picking tool) is shown. An example of the optical microscope photograph of MG63 on the culture dish collected in the HA compact is shown. The graph which shows a time-dependent change of the number of MG63 extract | collected by a HA molded object is shown. An example of the optical microscope photograph of the MG63 cell seed | inoculated by the HA molded object which extract | collected MG63 is shown.

Claims (3)

By placing a cell picking tool consisting of a molded body having a cell-adhesive concave structure in a cell collection site in a cell culture vessel, cells are adhered to and grown on the molded body side, and passive cell collection is performed. A cell operation method comprising handling and operating a picking tool together,
(1) The molded body is a calcium phosphate ceramic.
(2) The molded body has a shape having a cross section with an aspect ratio (major axis / minor axis) of 1.005 to 5, and a part of an opening of a pore, a through hole, or a dimple when left on a flat surface Or all face down,
(3) concave structure is composed of a material having a cellular contact adhesion,
( 4 ) The opening area of the concave structure is 100 to 9 × 10 ⁶ μm ² .
( 5 ) When the concave structure opening and the cell sheet contact, the concave structure opening and the cell sheet adhere.
It Ru is characterized by using the cell picking tool, a cell sheet which is two-dimensionally cultured in a culture vessel, without using a cell dispersing agent, remains of the sheet, taken while keeping the coupling state of the cell A cell manipulation method characterized by the above . One type selected from the group of spherical shape, bead shape, lump shape, plate shape, polyhedron shape, corrugated shape, dendritic shape, and protruding shape, wherein the molded body is 5 × 10 ⁻⁴ to 1 × 10 ³ mm ³ Or the cell manipulation method of Claim 1 which is a 2 or more types mixed molded object. The molded body is selected from the group consisting of pores, through-holes, dimples, slits, nodes formed by protrusion-bonding portions, surface adsorbed proteins, hydrophilic surfaces, polymer coats, and oxide coatings, or two or more kinds The cell manipulation method according to claim 1, which is a molded body having a structure.